Reexamination of the binding site for pyridoxal 5'-phosphate in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum.
The high specificity of pyridoxal 5'-phosphate (PLP) for an essential lysyl residue of ribulosebisphosphate carboxylase/oxygenase was confirmed, but half-of-sites reactivity was not observed in contrast to an earlier report [Robison, P. D., Whitman, W. B., Waddill, F., Riggs, A. F., & Tabita, F. R. (1980) Biochemistry 19, 4848-4853]. Subsequent to reduction with [3H]borohydride and tryptic digestion of the enzyme inactivated by PLP, the sole labeled peptide was purified by successive chromatography on DEAE-cellulose, SP-Sephadex, and Sephadex G-25. The peptide, recovered in good yield, appeared essentially homogeneous by amino acid analysis, peptide mapping, and sequencing. Automated Edman degradation established the peptide's sequence as Val-Leu-Gly-Arg-Pro-Glu-Val-Asp-Gly-Gly-Leu-Val-Val-Gly-Thr-Ile-Ile-(PLP)Lys -Pro-Lys instead of Ala-Leu-Gly-Arg-Pro-Glu-Val-Asp-(PLP)Lys-Gly-Thr-Leu-Val-Ile-Lys as reported by Robison et al. (1980) [Robison, P. D., Whitman, W. B., Waddill, F., Riggs, A. F., & Tabita, F. R. (1980) Biochemistry 19, 4848-4853]. The sequence -Ile-Lys-Pro-Lys- in the former is identical with that encompassing Lys-175 in the carboxylase/oxygenase from spinach, which reacts preferentially with PLP and two other affinity labels. This finding of homology greatly strengthens the supposition that Lys-175 in the spinach enzyme and the corresponding lysyl residue in the Rhodospirillum rubrum enzyme are active-site residues and furthermore increases the likelihood of their functionality in catalysis.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002262:Carboxy-Lyases / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D010105:Oxygenases / Q000378:metabolism*; D010455:Peptides / Q000032:analysis; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism*
D000596:Amino Acids; D010455:Peptides; D011732:Pyridoxal Phosphate; D010105:Oxygenases; D002262:Carboxy-Lyases; D012273:Ribulose-Bisphosphate Carboxylase